Pathological prognostic assays are used to provide information to help guide and develop treatment regimes and predict outcomes for a myriad of cancer types. Early detection and accurate determination of the molecular basis of a cancer is a key feature in treating cancer patients. For many cancers, this requires multiple separate preparations of tissue samples from the patient to determine different morphological and molecular factors.
Typically, cancer samples are pathologically examined by fixing the cells onto microscopic slides and staining them using a variety of staining methods (e.g., morphological or cytogenetic stains). Stained specimens are then evaluated for the presence or absence of abnormal or cancerous cells and cell morphologies. Although providing only general information, histological staining methods are the most common methods currently practiced for the detection of cancerous cells in biological samples. Other staining methods often used for cancer detection include immunohistochemistry and activity stains. These methods are based on the presence or absence of specific antigens or enzymatic activities in cancerous cells. Other methods of detecting cancerous cells utilize the presence of chromosomal aberrations in cancer cells. In particular, the deletion or multiplication of copies of whole chromosomes or chromosomal segments, and higher levels of amplifications of specific regions of the genome are common occurrences in cancer. Chromosomal aberrations are often detected using cytogenetic methods such as Giemsa-stained chromosomes (G-banding) or fluorescent in situ hybridization (FISH).
Typically, biological samples stained by any of the aforementioned methods are manually evaluated by either a laboratory technician or a pathologist. Microscopic slides are viewed under low magnification to locate candidate areas and those areas are viewed under higher magnification to evaluate the presence of cancerous cells. Further, current methods usually require a single staining method at a time, and if more than one staining method is performed, it is usually not on the same exact cells. This adds to the chance of either false negative results associated with cytological staining methods or false positive results associated with immunogenic or activity-based staining methods. The inability to directly associate objective measures of morphology with particular genetic rearrangements when separate slides are used has limited usefulness of combining such measurements in a meaningful way.
In men, prostate cancer is the most prevalent form of cancer for all races. While each year over 300,000 men are diagnosed with prostate cancer in the U.S. alone, the currently available tests are notoriously inaccurate and subjective. As a result many incidences of prostate cancer are undiagnosed until the disease has progressed to late stages, including metastases. Both the incidence of prostate cancer and its associated mortality have been increasing over the past ten years. The clinically evident disease represents only the tip of the iceberg in that nearly 30 percent of all men over age 50 harbor a silent microscopic form of latent prostate cancer. Early detection methods currently in use are increasing the identification of this latent form of cancer, which now represents more than 11 million cases within the male in the United States. Growth rate studies indicate that these tumors appear to grow very slowly and that the great majority should remain clinically silent. It is estimated that about 50-65% of prostate cancer is localized, 9-17% has spread to an area near the prostate, and 20-25% has metastasized to other parts of the body.
The screening for prostate cancer is primarily by PSA (a blood test for Prostate Specific Antigen) and DRE (Digital Rectal Exam) testing. Confirmation of cancer is made by examination of tissue samples derived from needle biopsies. These methodologies cannot differentiate between benign disease and cancer. The failure to differentiate can result, for example, in exposure of patients with benign disease to treatments that are unnecessary and have side effects (e.g., impotence and incontinence). At present, factors to be considered in assessing cancer progression are estimates. Tumor volume, pre- and post-operative histological grading of cancer and high grade intraepithelial neoplasia, clinical and pathological tumor staging, and serum PSA may be employed to predict the biological aggressiveness of prostate cancer. Unfortunately, these techniques generally have only marginal predictive value. Moreover, it is estimated that PSA testing misses 20%-30% of all individuals with cancer. Accordingly, there is a clear need for diagnostics with better sensitivity and specificity.
It is well accepted that the epigenetic and genetic transformation of a normal prostatic cell to a cancer cell with progression to a metastatic phenotype requires multiple steps. The development of methods to identify these changes in order to better select therapies and to predict tumor aggressiveness has been the subject of much work in prostate cancer. In spite of the progress made in evaluating the progression of prostate cancer, it is evident that improvements are needed in the accuracy of such determinations.
Thus, there is a widely recognized need for, and it would be highly advantageous to have, a method of analyzing cancer and cancer-associated morphologies that can analyze multiple-variables in single cells of a biological sample within a single acquisition, providing a higher confidence level for identification of specific mechanisms that drive the prognosis of cancer, and providing more information to the health care professionals in the designing and selecting of treatment protocols.